节点文献
芎芍胶囊对动脉粥样硬化兔胆固醇逆向转运及炎症反应的影响
【作者】 张艳虹；
【导师】 徐凤芹；
【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2014， 博士
【摘要】 目的：观察芎芍胶囊对动脉粥样硬化(AS)兔血管壁AS形成的影响,从促进主动脉胆固醇跨膜转运、肝脏胆固醇代谢影响胆固醇逆向转运(RCT)过程,抑制炎症反应及保护高密度脂蛋白(HDL)抗炎作用等方面探讨活血化瘀中药抗AS的新机制。实验方法和结果：60只新西兰兔随机分为6组：空白对照组,模型组,芎芍小、中、大剂量组,辛伐他汀组。采用单纯高脂喂养法复制兔AS模型,分别在给药前,给药6周、12周及15周末4个时间点经耳缘静脉采血,15周末麻醉后处死动物,取胸主动脉及肝脏。肉眼观察大体标本各组兔脂纹、脂斑情况,HE染色观察各组兔AS斑块形成情况,油红O染色观察各组兔动脉壁脂质沉积情况；酶联免疫吸附法(ELISA)测定血清白介素-2(IL-2)、肿瘤坏死因子-α(TNF-α)、髓过氧化物酶(MPO)、对氧磷酶1(PON1)水平,测定动脉血管平滑肌细胞内总胆固醇(TC)、游离胆固醇(FC)水平,肝脏细胞内肝X受体α(LXRα)水平；凝胶迁移实验(EMSA)测定核因子·-κB (NF-κB)p65活性；蛋白印迹法(WB)测定主动脉壁内小凹蛋白-1(cav-1)、亲环素A表达量,肝脏B类Ⅰ型清道夫受体(SB-B Ⅰ)表达量；实时定量聚合酶链式反应(real-time PCR)测定主动脉壁cav-lmRNA、亲环素A mRNA、NF-κB mRNA表达量,肝脏SR-BI mRNA, LXRαmRNA表达量结果显示：空白对照组兔主动脉管壁表面光滑,内皮细胞连续完整,无脂质沉积；模型组兔主动脉管壁满布脂质斑块,平滑肌层细胞内见大量脂质沉积；芎芍小、中、大剂量组随用药剂量增加,管壁表面脂纹逐渐减少,显微镜下见形成斑块逐渐减少,管壁细胞内脂质沉积减少；其中芎芍大剂量组与辛伐他汀组在大体标本及HE染色、油红O染色结果上无明显差异。15周末模型组兔平滑肌细胞内TC较空白对照组明显升高(P=0.006,P<0.01),FC呈现相同的趋势(模型组与空白对照组比P=0.008,P<0.01)。芎芍中剂量组较模型组细胞内TC明显减少(P=0.035,P<0.05),芎芍大剂量降低效果更加明显(P=0.004,P<0.01)。芎芍小剂量组较模型组FC明显减少(P=0.024,P<0.05),芎芍中、大剂量组(P=0.001,P<0.01)及辛伐他汀组FC水平降低更明显(P=0.006,P<0.01),模型组兔cav-1表达量明显下降(P=0.001,P<0.01),芎芍中、大剂量组较模型组表达增加(P=0.022<0.05,P=0.002<0.01),辛伐他汀组及芎芍小剂量组与模型组相比cav-1表达也有增加趋势,但无统计学意义(P=0.063,P=0.179)。模型组兔亲环素A表达量较对照组增加(P=0.013,P<0.05),芎芍中、大剂量组及辛伐他汀组较模型组亲环素A表达减少(P=0.017<0.05,P=0.006,0.000<0.01)。与对照组相比,模型组兔明显下降(P=0.001,P<0.01),辛伐他汀组及芎芍大剂量组较模型组增加cav-lmRNA(P=0.014,P<0.05；P=0.001,P<0.01)。模型组兔亲环素表达量与对照组相比AmRNA有下降趋势,但无统计学差异(P=0.884,P>0.05)；芎芍大剂量组及辛伐他汀组与模型组相比明显增加(P=0.003,P=0.004<0.01),余治疗组较模型组无明显差异。肝细胞测定结果显示,模型组兔LXRα表达量较空白对照组下降(P=0.798,P>0.05),但没有统计学差异。芎芍大剂量组较模型组表达增加(P=0.005,P<0.01),余治疗组与模型组相比表达有增加趋势,但无统计学意义。模型组LXRamRNA较空白对照组表达量明显增加(P=0.000,P<0.01),各治疗组较模型组无明显差异(P>0.05),模型组与空白对照组兔相比肝脏SR-BI蛋白表达量有减少趋势,但无统计学差异(P=0.281,P>0.05),而芎芍胶囊各治疗组较模型组SR-B Ⅰ蛋白表达量有增多趋势,无统计学差异。辛伐他汀组兔与模型组相比,SR-BI蛋白表达量增多明显(P=0.049,P<0.05),其与芎芍大剂量组相比表达量无明显差异。模型组兔SR-BI基因表达量较空白对照组明显增加(P=0.021,P<0.05)。各给药组与模型组相比,基因转录水平无明显增多(P>0.05)。芎芍中剂量组、大剂量组及辛伐他汀组兔SR-BI基因转录水平有升高趋势,且大剂量组较中剂量组更加明显。炎症因子方面,对4个不同时间点的测量结果显示,造模及给药时间长短、不同治疗方法均对各组兔IL-2、TNF-α水平产生影响。模型组与空白对照组相比IL-2水平增加,但无统计学差异(P=0.790,P>0.05),与模型组相比,芎芍中、大剂量组及辛伐他汀组兔血浆IL-2水平均明显降低,有统计学意义(P=0.004<0.01,P=0.026,P=0.013,P<0.05)。模型组兔TNF-α水平较空白对照组升高,但无统计学差异(P=0.120,P>0.05),芎芍中、大剂量组及辛伐他汀组较模型组兔TNF-α水平明显降低(P=0.032,P=0.013,P=0.049,P<0.05)。模型组比空白对照组兔主动脉NF-κBp65表达量增多,但并无统计学差异(P=0.169,P>0.05),各给药组与模型组相比蛋白表达量减少,其中芎芍大剂量组(P=0.035,P<0.05),辛伐他汀组(P=0.001,P<0.01)与模型组相比下降更明显。模型组NF-κBmRNA表达量与空白对照组相比略有减少,但无统计学差异(P=0.893,P>0.05),芎芍大剂量组表达量较模型组增加(P=0.031,P<0.05),各给药组基因表达量与空白对照组无明显差异(P>0.05)。各治疗组兔PON1水平无明显差异(P>0.05),模型组兔与空白对照组兔血浆PON1亦无差异(P=0.697,P>0.05)。模型组兔血浆MPO水平与空白对照组相比明显升高(P=0.025,P<0.05),芎芍中剂量组及大剂量组、辛伐他汀组较模型组血浆MPO水平明显下降(P=0.028,P=0.013,P<0.05；P=0.004,P<0.01),芎芍中、大剂量组与辛伐他汀组间比较无差异护>0.05)。结论：1.芎芍胶囊能够抑制AS兔主动脉斑块形成,减少动脉壁细胞内脂质沉积。2.芎芍胶囊能够通过保护AS兔主动脉平滑肌细胞内cav-1蛋白表达,增加主动脉细胞内胆固醇跨膜转运促进细胞内胆固醇流出,从而减少主动脉细胞内TC及FC含量,起到抗AS作用。3.芎芍胶囊能够促进AS兔肝脏表达LXRα,促进肝脏内胆固醇代谢,推动RCT过程,其对SR-BI促进作用不明显。4.芎芍胶囊能够降低AS兔血浆IL-2、TNF-a水平,降低主动脉细胞核内NF-κBp65的活性水平,抑制AS中的炎症反应。5.芎芍胶囊能够降低AS兔血浆中MPO水平,保护HDL抗炎功能,维持其正常运载脂质的作用,其对血浆PON1水平作用不明显。
【Abstract】 Objective:To observe the effects of Xiongshao Capsule(XSC) on plaques of rabbit models of atherosclerosis(AS) and investigate the new mechanisms of traditional Chinese medicines for invigorating blood circulation and eliminating stasis against AS through promotion of transmembrane transport of cholesterol in aorta, effect of cholesterol metabolism in liver on reverse cholesterol transport(RCT), inhibition of inflammatory response and protection of the anti-inflammatory action of high density lipoprotein(HDL).Experimental methods and results:There were60New Zealand rabbits divided into six groups randomly:blank control group, model group, XSC low, middle, and high dose group, and simvastatin group. The rabbits of AS were established by simple high-fat feeding method. The blood samples were collected from ear marginal veins before and after drug administration for6weeks,12weeks, and15weeks. The thoracic aortas and livers of the animals were taken after execution under anesthesia at the end of15weeks. The rabbit fatty streaks and fatty plaques of gross specimen were inspected by visual study. The formation of rabbit arterial plaque was observed by HE staining. The lipidosis in arterial wall was viewed by oil red o staining.The levels of interleukin-2(IL-2),tumor necrosis factor-a (TNF-a), myeloperoxidasein(MPO) paraoxonase-1(PON1) in serum were measured by enzyme-linked immuno sorbent assay(ELISA). The total cholesterol(TC), free cholesterol(FC) in vascular smooth muscle cell and liver X receptor a(LXRa) in livers were determined. The activity of nuclear factor-κBp65(NF-KBp65) was tested by electrophoretic mobility shift assay(EMSA). The caveolin-1(cav-1) and expressions of cyclophilin A(CyPA) and scavenger receptor claBs B type I (SR-B I) in liver were measured by western blot(WB). The expressions of mRNA of cav-1mRNA, CyPA and NF-κB in arterial wall, mRNA of SR-B I in liver and mRNA of LXRawere evaluated by real-time quantitative polymerase chain reaction(real-time PCR).Results:The surface of aortic wall in blank control group is smooth and the endothelial cells are continuous without lipidosis. The aortic wall in model group is covered with lipid plaques, in which plaques and lipidosis in smooth muscle cells can be found obviously. The fatty streak in the surface of aortic wall, formation of plaques under microscope, and lipidosis in the cells of aortic wall are declining with the increasing administration of drug in XSC low, middle, and high dose group. There are no differences in gross samples, results of HE staining and red oil0staining between XSC high group and simvastatin group.TC in smooth muscle cells in model group is significantly higher than that in blank control group at the end of15weeks(P=0.006,P<0.01), while FC shows the same trends in both groups(P=0.008,P<0.01). TC in cells in XSC middle dose group is significantly lower than that in model group(P=0.035,P<0.05), which is even much lower in XSC high dose group, compared to model group(P=0.004,P<0.01). FC in XSC low dose group is significantly lower than that in model group(P=0.024,P<0.05), and it is even much lower in XSC middle and high dose group and simvastatin group, compare to model group(P=0.001,P<0.01,P=0.006,P<0.01, respective ly). The expression of cav-1in model group is significantly declining (P=0.024,P<0.05), but it is improved in XSC middle and high dose group(P=0.001,P<0.01). Com pared to model group, the expression of cav-1in simvastatin and XSC low dose groups presents a trend of increasing, which shows no statistical signify cance(P=0.063,P=0.179).The expression of CyPA in model group is higher than that in control group (P=0.013,P<0.05), while those in XSC middle and high dose group and simvastatin group are lower than that in model group(P=0.017<0.05,P=0.006,0.000<0.01,respectively). Compared to control group, the cav-1mRNA in model group is significantly lower(P=0.001,P<0.01) but higher in simvastatin group and XSC middle and high group(P=0.014,P<0.05,P=0.001,P<0.01, respectively). Compared to control group, the expression of CyPA mRNA in model group presents a downward trend, which has no statistical significance (P=0.884,P>0.05). And the expressions of CyPA mRNA in XSC high dose and simvastatin groups are higher than that in model group(P=0.003,P=0.004, respectively, both P<0.01), while there is no difference between other treatment groups. The test results of liver cells show that the expression of LXRa in model group declines slightly than that in blank control group, which shows no statistical significance(P=0.798,P>0.05). And compared to model group, the expression in XSC high dose group presents an upward trend(P=0.005,P<0.01) and other treatment groups show the same trend without statistical significance. The expression of LXRa in model group is significantly higher than that in blank control group(P=0.000,P<0.01) and there is no significant difference between each treatment group and model group (P>0.05).Compared with blank control group, the protein expression of SR-B I in model group presents a down ward trend, which has no statistical signi ficance(P=0.281,P>0.05). Compared to model group, the protein expressions of SR-B I in XSC low, middle and high dose groups show an upward trend, which doesn’t show statistical significance, either. The expression in simvastatin group is significantly higher than that in model group(P=0.049,P<0.05), while there is no difference between simvastatin group and XSC high dose group. The gene expression of SR-B I in model group is significantly higher than that in blank control group(P=0.049,P<0.05). There is no significantly difference in the level of gene transcription between each treatment group and model group(P>0.05).The gene transcription levels of SR-B I in XSC middle and high dose groups and simvastatin group show an upward trend, and XSC high dose group is especially more obvious than XSC middle dose group.In the matter of inflammatory factors, the measurement results from the4different time points show that modeling, time for the drug administration, and different treatment methods all have effects on the levels of IL-2and TNF-a in each group. Compared to model group, the serum level of IL-2in XSC middle and high dose groups and simvastatin group are significantly lower, in which there are statistical significances (P=0.004<0.01,P=0.026, P=0.013, respectively, both P<0.05). The level of TNF-a in model group is higher than that in blank control group, which has no statistical significance(P=0.120,P>0.05). The level of TNF-a in XSC middle and high dose groups and simvastatin group is significantly lower than that in model group(P=0.032,P=0.013,P=0.049, respectively, all P<0.05). Compared to blank control group, the expression of NF-κBp65in artery in model group is higher, which shows no statistical significance(P=0.169,P>0.05). The expression of protein in each treatment group is lower than that in model group, in which XSC high dose group(P=0.035,P<0.05) and simvastatin group(P=0.001,P<0.01) are especially significant. The expression of NF-κB mRNA in model group is slightly lower than that in blank control group, which shows no statistical signify cance(P=0.893,P>0.05). The expression in XSC high dose group is higher than that in model group(P=0.031,P<0.05). There is no significant difference in gene expression between each treatment group and blank control group(P>0.05).No significant difference has been found in the level of PON1between each treatment group(P>0.05). There is also no difference in the serum level of PON1between model group and blank control group(P=0.697,P>0.05). The serum level of MPO in model group is significantly higher than that in blank control group(P=0.025,P<0.05). Compared to model group, the same decline in the serum level of MPO is also significant in XSC middle and high dose groups as well as simvastatin group(P=0.028,P=0.013,P<0.05,P=0.04,P<0.01, respectively). There is no differ rence between XSC middle and high dose group and simvastatin group(P>0.05).Conclusions:1. XSC can inhibit the plaque formation in artery of rabbits with AS and reduce lipidosis in artery wall cells.2. XSC can reduce the contents of TC and FC in artery cells through protecting the protein level of cav-1and increasing transmembrane transport of cholesterol in artery cells to promote cellular cholesterol efflux, which has an effect on anti-AS.3. XSC can promote the expression of LXRa, cholesterol metabolism in liver and the process of RCT, and it has no obvious effect on SR-B I.4. XSC can reduce the serum level of IL-2and TNF-a in rabbits with AS, and lower the activity level of NF-κBp65in artery cell nuclear, which inhibit the inflammatory response in AS.5. XSC can reduce the serum level of MPO in rabbits with AS, protect the anti-inflammatory function of HDL to maintain the normal lipid transport function and has no significant effect on PON1.